In this proposal we will use molecular genetic methods to dissect the function of Mrgs, and the circuitry and function of the neurons that express these GPCRs. Using knockout mice expressing fEGFP from the MrgA1 and MrgD loci, we will ask: 1) what are the peripheral and central projection targets of MrgA1+ and MrgD+ neurons?; 2) Do MrgA and MrgD play a role in the peripheral or central targeting of sensory axons?; 3) Do MrgA and/or MrgD exhibit allelic exclusion? In collaboration with Allan Basbaum, we will ask: 4) How are MrgA1 and MrgD expression regulated in chronic inflammation and nerve injury?; 5) What are the behavioral consequences of loss of MrgA and MrgD function on nociception? In collaboration with Mel Simon, we will ask 6) How do ligands for MrgA affect the physiology of MrgA-expressing neurons in vitro, and are these effects dependent on MrgA function? In collaboration with Basbaum, we will ask whether deletion of the receptors affects the behavioral response to MrgA ligands in vivo. Using a tet-dependent transcriptional activator (rtTA) expressed from the MrgA and MrgD loci, and a series of tet-inducible responder mice, we will map the second-order neurons that receive afferent input from MrgA- and MrgD-expressing neurons, and third- or higher-order neurons to which they project. In collaboration with Basbaum, we will additionally investigate 2) the behavioral consequences of killing MrgA- and MrgD-expressing neurons at different stages of postnatal development, and 3) the behavioral consequences of reversibly silencing MrgA- and MrgD-expressing neurons. We will generate similar targeted mutations in MrgC11 and MrgB4/5, and ask an analagous series of questions about the function of these genes and of the neurons and circuits in which they are expressed. Using MrgC11 and MrgA knockout mice, we will delete the MrgAC gene complex and examine its functional consequences. Finally, we will exploit the specificity of MrgD expression to compare the gene expression profiles of isolated MrgD+, IB4+ and MrgD-, IB4+ nociceptive neurons. These studies may identify 1) additional genes involved in MrgD-related signaling; and/or 2) markers for other subpopulations of IB4+ neurons will different end-organ specificities. Together, these experiments should provide new insight into molecular and cellular mechanisms of nociception, and may ultimately lead to the development of novel pain therapeutics.